Abstract

In order to combat a wide array of pathogens, the immune system must be able to generate cells with the ability to recognise
virtually any pathogen while at the same time balancing this diversity with the maintenance of sufficient numbers of cells
specific for each pathogen in order to mount an effective response. T cells, which are important for both the direct elimination
of infected cells as well as providing support for other immune responses to pathogens, generate immunologic diversity by
imprecise joining of gene segments to generate either α/β or γ/δ heterodimeric receptors during development. Although the
potential diversity of rearranged T‐cell receptors (TCR) is many orders of magnitude greater than the number of T cells in any given individual, highly regulated steps in development,
as well as restrictions in the specificity of TCR, facilitate the generation of sufficient numbers of T cells with each TCR to ensure efficacy in pathogen control.

Key Concepts

The T‐cell repertoire must balance diversity of antigen recognition with sufficient numbers of each specificity to elicit
protection.

T cells may express either αβ or γδ T‐cell receptors, which have different roles in immunity.

Figure 1. Somatic recombination of semi‐randomly selected germline encoded TCR genes is required to form the variable regions of TCRs. T‐cell receptor genes can make rearrangements that are either productive or nonproductive, and rearrangements can be attempted on both copies of each locus. The β‐ and δ‐chain variable regions are composed of V, D and J segments, the first rearrangement joins a D→J, and the second rearrangement joins a V→DJ. The α‐ and γ‐chains are composed of only V and J segments and therefore require only a single V→J rearrangement. While the β and γ genes are located discreetly on chromosome 7, the δ segments are interspersed within the α locus on chromosome 14. Finally, successive gene rearrangements allow the replacement of one TCR chain by another. The multiplicity of V, D and J gene segments allows successive rearrangement events to occur if an unproductive rearrangement leads to an inadequate receptor chain. This process may continue until either a productive rearrangement occurs or the supply of gene segments is exhausted.

Figure 2. Somatic recombination utilises some of the enzymes and mechanisms used universally in DNA recombination and repair; however, there are also enzymes and mechanisms specific to cut and rejoin DNA to rearrange TCR genes. The recombination of V, D and J gene segments is directed by sequences referred to as recombination signal sequences (RSS), which flank the 3′ side of V segments, both sides of D segments and the 5′ side of J segments. There are two types of RSSs, one containing a 12 nt spacer and one with a 23 nt spacer (corresponding to one and two turns of the double helix, respectively). Recombination of VDJ genes can only occur between genes flanked by different RSSs (‘12/23 rule’). Both RSSs are composed of their respective 12 or 23 nt spacer flanked on either side by a 7 nt heptamer and 9 nt nonamer. In addition to their function of providing recognition sites for enzymes to cut and rejoin the DNA, RSSs also ensure that correct joining of gene segments occurs (i.e. D→J followed by V→DJ for TCRβ/δ or V→J for TCRα/γ).

Figure 3. Taking place in the thymus, T‐cell development is a highly ordered process of events occurring at distinct stages of cell development. Upon receiving NOTCH signalling in the bone marrow, common lymphoid progenitor cells travel to the thymus and enter through the high endothelial venule (HEV). Referred to at this point as double‐negative 1 (DN1) cells (lacking expression of either CD4 or CD8 coreceptors), these early progenitors begin β‐, γ‐ and δ‐chain gene rearrangements. At the DN2 stage, the cells migrate towards the cortex and complete β‐, γ‐ and δ‐chain rearrangements. If a successful γδ rearrangement occurs before a successful β‐chain is formed, the cells will express γδ TCRs and exit the thymus as mature γδ T cells, forgoing any further selective processes. If productive β‐chain rearrangement happens first, the β‐chain will be paired with a surrogate α‐chain (pTα) as the cell moves into the DN3 stage. At this point, the cell will assess the β‐chain rearrangement (β‐selection), and should productive rearrangement have occurred, the cell will enter the DN4 stage and proliferate to maximise on the success of the productive β‐chain rearrangement. In the early double‐positive (DP) stage, the cells will undergo α‐chain rearrangement. Upon successful α‐chain VJ recombination, the cell will shuttle its αβ TCR to the cell surface, upregulate both CD4 and CD8 coreceptors and undergo a positive selection process to ensure self‐MHC restriction mediated by the cortical epithelial cells of the thymus. Depending on which MHC (I or II) the cell identifies with, it will downregulate the other coreceptor and become either a single‐positive (SP) CD8 or CD4 T cell. At the SP stage, the cell migrates back towards the corticomedullary junction and undergoes its final selective process (negative selection) carried out by medullary epithelial cells, to delete any self or autoreactive cells. After negative selection, the newly matured naïve T cell will leave the thymus back through the HEV and exit to the periphery in search of its cognate antigen.

Figure 4. The αβ T‐cell receptor (αβTCR) is a dimeric structure composed of two type‐I glycoprotein chains linked together by disulphide bridges. Each chain is composed of two extracellular domains (variable and constant), a hydrophobic transmembrane region and a very short intracytoplasmic region. Owing to the short nature and lack of any intracellular protein docking sites or signalling motifs, signal transduction following antigen recognition is mediated by a multimeric complex referred to as CD3. The three CD3 dimers (ϵγ, δϵ and ζζ) contain acidic transmembrane residues that interact with basic residues on the transmembrane portion of the TCR chains. Signal transduction is mediated by intracellular tyrosine activation motifs (ITAMs) contained on the intracellular portions of the CD3 subunits. The γ, δ and ϵ subunits each carry one ITAM, while the ζ subunits contain three. Once phosphorylated upon TCR activation, the ITAMs will recruit several tyrosine kinases such as ZAP70, triggering a signalling cascade leading to T‐cell activation and/or effector function.

Figure 5. Structural reorganisation of cell surface proteins upon antigen recognition and activation. Before antigen recognition occurs, TCRs, coreceptors, adhesion molecules and signalling components are somewhat evenly distributed across the cell surface (top). In the course of T‐cell activation, however, a very specific reorganisation of cell surface proteins takes place to form the immunological synapse (IS). This reorganisation results in different clusters of proteins relative to the centre of the synapse. Referred to as supramolecular activation clusters (SMACs) and named relative to their location from the IS, each SMAC is composed of distinct proteins in a way that allows optimal signalling, costimulation and adhesion. The most central cluster, referred to as the central SMAC (c‐SMAC), is composed of TCR/CD3 complexes, CD4/8 coreceptors, CD28 costimulatory molecules and LAT signalling proteins. The intermediate cluster, referred to as the peripheral SMAC (p‐SMAC), is composed mainly of adhesion proteins such as LFA‐1 and ICAM‐1. Finally, the most distal cluster, referred to as the distal SMAC (d‐SMAC), is composed of the long CD45 phosphatases.

Corthay
A
,
Nandakumar
KS
and
Holmdahl
R
(2001) Evaluation of the percentage of peripheral T cells with two different T cell receptor alpha‐chains and of their potential role in autoimmunity. Journal of Autoimmunity
16: 423–429.

Marche
PN
and
Kindt
TJ
(1986) Two distinct T‐cell receptor alpha‐chain transcripts in a rabbit T‐cell line: implications for allelic exclusion in T cells. Proceedings of the National Academy of Sciences of the United States of America
83: 2190–2194.

Mason
D
(1998) A very high level of crossreactivity is an essential feature of the T‐cell receptor. Immunology Today
19: 395–404.

Murugan
A
,
Mora
T
,
Walczak
AM
and
Callan
CG
Jr
(2012) Statistical inference of the generation probability of T‐cell receptors from sequence repertoires. Proceedings of the National Academy of Sciences of the United States of America
109: 16161–16166.